Chromosomal translocations that affect the proto-oncogene MLL (Mixed Lineage Leukemia) occur in aggressive human acute leukemias, both in children and adults. Fusion of MLL to one of more than 60 partner genes results in generation of the MLL fusion oncoprotein which upregulates expression of Hox genes required for normal hematopoiesis, and ultimately leads to the development of leukemia. Patients harboring fusion of the MLL gene suffer from very aggressive leukemias and respond poorly to available therapies, with only ~35% five-year survival rate, emphasizing that novel therapeutic treatments are urgently needed. All oncogenic MLL fusion proteins have a preserved N-terminal fragment of MLL that interacts with menin, a protein encoded by MEN1 (Multiple Endocrine Neoplasia 1) gene. Importantly, the interaction of menin with the MLL fusion proteins is critical to the leukemogenic activity of the MLL fusions. Therefore, menin functions as an essential oncogenic cofactor in MLL leukemias, and represents a valuable molecular target for therapeutic intervention. We hypothesize that direct targeting of the interaction between menin and MLL fusion proteins with small molecules will reverse the oncogenic potential of MLL fusions and will inhibit the development of leukemia. By applying two independent high throughput screens, we identified over 20 small molecules which specifically bind to menin and inhibit the menin-MLL interaction. These compounds, which belong to several distinct chemotypes, represent the first small molecule inhibitors targeting this interaction. Applying a combination of biochemical, biophysical and cellular assays we were able to evaluate their potency, specificity, and mechanism of action. The most potent compounds competitively displace the MLL-derived peptide from menin with the IC50 values at low micromolar range, with the most potent MI-1 and MI-2 representing two different scaffolds. These compounds selectively inhibit the growth of leukemia cells with MLL translocations, and induce apoptosis and differentiation of MLL leukemia cells. We have tested analogues of MI-1 and MI-2 to establish an initial structure-activity relationship and improve their potency. The most potent analogue of MI-1 synthesized in our laboratory has IC50 = 430 nM for inhibition of the menin-MLL interaction. This compound selectively inhibits the cell growth, induces apoptosis and differentiation of MLL leukemia cells, substantially downregulates expression of Hoxa9 and Meis1 genes, and inhibits transformation by MLL fusion oncoprotein, demonstrating a highly specific mode of action. In this project we propose to further optimize these lead compounds by combining medicinal chemistry, NMR, biochemical and biophysical methods. Compounds will be tested for their activity in leukemia cells and on normal bone marrow cells to validate their mechanism of action and exclude compounds which might cause toxicity issues. In vivo efficacy studies in mouse models of MLL leukemia will also be pursued for selected compounds. If successful, our studies will result in compounds which may provide a novel therapeutic approach for the treatment of leukemias with MLL translocations. PUBLIC HEALTH RELEVANCE: We are proposing to develop small molecules that may reverse the oncogenic function of MLL fusion proteins in acute leukemias. Ultimately, such compounds could be used as highly specific drugs for treatment of patients with aggressive forms of leukemia, which poorly respond to current therapies.